Production of penicillin



Patented Sept. 23, 1952 PRODUCTION OF PENICILLIN Erling Knud Frederiksen, Holte, and Erling Juhl Nielsen, Charlottenlund, Denmark, assignors, by mesne assignments, to American Cyanamid Company, New York, N. Y., a firm NoQ-Djrawing. Application May 3, 1949, Serial No.

91,225. In Denmark May 5, 1948 7 1 Claim. (01. 19536) The present invention relates to the produc tion of penicillin.

The penicillin molecule contains a group, which is different for different kinds of penicillin; and

which is signified by R in the penicillin formula below:

When cultivating penicillin-producing microorganisms in submerged cultures, different kinds of penicillin are formed, among which the one named G-penicillin has the greatest therapeutic activity. The group R in the G-penicillin is the benzyl group CeH5CH2-. If cultivated in surface cultures there are also obtained different kinds of penicillin with X-penicillin as the most active kind. In X-penicillin R is a p-oxybenzyl group: HOCsH4CI-I2. In other known penicillins R. signifies other organic groups, generally aliphatic groups.

It is known that the yield of penicillin may be increased, especially by submerged cultures, by adding assisting substances-so called precursors-which contain the desired group R, whereby it is possible to increase somewhat the re ative contents of the desired kind of penicillin, especially G-penicillin. As a precursor phenylacetic acid, its salts or its esters have been used.

The use of said precursors is, however, not without disadvantages because they are to a certain degree toxic to the microorganism used for producing the penicillin, and it has, therefore, been necessary to add these compounds by degrees as they are used during the cultivation, which involves extra work, extra control and infection.

risks. Furthermore these compounds are to a considerable degree soluble in the organic solvents used for extracting the free penicillin and it is therefore difiicult to remove the inevitable surplus of said compounds from the penicillin, at least during the primary purification.

We have found that the above disadvantages do not apply to all kinds of esters, and it is an object of our invention to produce penicillin by a process, in which the above disadvantages are avoided. Particularly the purpose of the invention is to provide for a method of producing penicillin using as a precursor esters of phenylacetic acid which are substantially non-toxic to the microorganisms used. Another purpose of the invention is to provide for a precursor, which is not extracted by the'solvent' used for extracting penicillin from the developed cultures.

Generally this is obtained according to the invention by usingas precursors in the-production of penicillin certain phenylacetic acid esters which are relatively non-toxic to the penicillin producing organisms, and which, moreover, may form salts not soluble in the solvents used for extracting the penicillin.

With these general statements of the objects and purposes of our invention we will now proceed to describe the embodiment thereof and the manner in which ourinvention is carried out, and it will be understood that while we have described what may be considered as a preferable embodiment of our invention, we do not limit ourselves to the precise conditions or proportions herein set forth, as they may be varied by those skilled in the art in accordance with the particular purposes for which they are intended, and the conditions under which they are to be utilized.

According to the invention there is used as a precursor in the production of penicillin an ester of phenylacetic acid with a nitrogen containing alcohol. As such esters are far less toxic to the penicillin forming microorganisms than the hitherto used compounds, it is possible, if desired, to add at once to the culture the whole amount of precursor necessary to obtain the desired result. Such esters have further the advantage that they form salts, which remain in the water phase when extracting the penicillin with organic solvents. This is why intermediary products free from the precursor are more rapidly obtained.

We have found that esters of phenylacetic acid with aminoalcohols containing tertiary linked nitrogen, for instance diethylamino ethanol, the salts of which are practically insoluble in the solvents generally used for extracting the penicillin, are particularly suitable. Also esters With amino alcohols, in which the nitrogen is ringlinked, for instance in a piperidine group, as in the case of 2(1-piperidyl) ethanol, are suitable.

Both the relative non-toxicity and the said circumstances of solubility are remarkable. Many esters of phenylacetic acid, for instance the ethyl ester, are at least as detrimental as phenylacetic acid itself or even more so, and their solubility does not prevent them in accompanying the penicillin during part of the purification processes. Esters of aminoalcohols with primary or secondary linked nitrogen possess to a lesser degree the desired properties characterizing the esters of amino alcohols with tertiary linked nitrogen, but

may be used advantageously instead of phenylacetic acid itself when foregoing the addition of the whole amount of precursor at once.

Our method is illustrated by the following example, which in no way is to be considered restrictive.

Example 300 kilos cornsteep liquor, 80 kilos lactose, 40

kilos calcium carbonate and 4000 liters of water are autoclaved and inoculated with a suitable culture of Penicillium notatwm or P. chrysogenum and then fermented in a..closed vessel, whileaerating vigorously. In such medium after: 60-70 hours there are produced about 300 units penicillin per ml., of which about 90 per. cent is Krpenie cillin, which is only slightly active;

When adding about 800 grains of the ester of phenylacetic acid with diethylanf ino ethanol be I per liter without detriment to the micro-organism. Having thus described our invention, we claim: A method for the production of penicillin which comprises the step of cultivating a penicillin producing fungus of the genus Penicillium in an aqueous nutrient solution containing-the: phenylacetic aoidester of dietliylamino ethanol, said ester being present in the nutrient solution in an amount not exceeding 2 grams per liter of solution.

ERLING KNUD FREDERIKSEN. ERLING J UHL NIELSEN.

REFERENCES CITED The following references are of record in the file of this patent:

UNITED STATES PATENTS Number Name. Date 20 2,423,873 Coghill et al July 5, 1947' 2,448,791 Foster et al-. Sept; 7;.1948' 2,479,297 Behrens Aug: 16,1949 23.532980- Woodruff et-ali Dec; 5,1950 

